SCANNING ALANINE MUTAGENESIS AND DE-PEPTIDIZATION OF A CANDIDA-ALBICANS MYRISTOYL-COA-PROTEIN N-MYRISTOLYTRANSFERASE OCTAPEPTIDE SUBSTRATE REVEALS 3 ELEMENTS CRITICAL FOR MOLECULAR RECOGNITION

Candida albicans produces a single myristoyl CoA: protein N-myristoylt ransferase (Nmt) that is essential for its viability. An ADP-ribosylat ion factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-termin al Arf sequence was used as the starting point to identify elements cr itical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of pepti des with single Ala substitutions at each position of GLYASKLS-NH2, es tablished that its Gly(1), Ser(5) and Lys(6) residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competiti ve for peptide (K-i = 15.3 +/- 6.4 mu M) and noncompetitive for myrist oyl-CoA (K-i = 31.2 +/- 0.7 mu M). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C terminal truncations, and (iii) manipulation of the physical-chemica l properties of its residues 1, 5, and 6, confirmed the important ster eochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser(5), and the epsilon-amino group of Lys(6). Remarkably, replacemen t of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminou ndecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-S KLS-NH2, that was 38-fold more potent (K-i = 0.40 +/- 0.03 mu M) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS- NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resul ted in 26- and 34-fold increases in IC50, confirming the important con tribution of the amine to recognition. Removal of Leu-Ser from the C t erminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide in hibitor with a K-i (11.7 +/- 0.4 mu M) equivalent to that of the start ing octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, ci s-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasiz ing the requirement for proper presentation of the hydroxyl group in t he dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibi tory activity > 100 fold, while deletion of the epsilon-amino group (N le) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L- Lys, producing a competitive dipeptide inhibitor with similar potency (K-i 11.9 +/- 1.0 mu m). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecan oyl-SH-NH2, establish that a simple alkyl backbone can maintain an app ropriate distance between three elements critical for recognition by t he fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. T hese compounds contain one peptide bond and two chiral centers, sugges ting that it may be feasible to incorporate these elements of recognit ion, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.